Vitro methods for screening for blood coagulation disorders using metal ions

ABSTRACT

In vitro methods for qualitative screening and/or quantitative determination of the functional activity of components of the Protein C anticoagulant pathway of blood coagulation are described. The methods entail measuring the conversion rate of a substrate by an enzyme, the activity of which is related to the Protein C anticoagulant activity, in a blood sample of a human comprising coagulation factors and said substrate, after at least partial activation of coagulation through the intrinsic, extrinsic or common pathway and triggering coagulation by adding calcium ions; and comparing said conversion rate with the conversion rate of a normal human blood sample determined in the same way. The methods include the addition of additional metal ions to the sample to enhance activity, sensitivity and resolution. Kits and reagents for use in the methods are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/050,441, filedJan. 16, 2002 now U.S. Pat. No. 6,730,490, which is incorporated hereinby reference and which is a continuation of U.S. Ser. No. 09/273,413,filed Mar. 19, 1999, now U.S. Pat. No. 6,395,501, which is incorporatedherein by reference and which claims priority to European PatentApplication Serial No. 98105043.8, filed Mar. 19, 1998.

FIELD OF THE INVENTION

The present invention relates to in vitro methods, kits and reagents forthe qualitative screening and quantitative determination of thefunctional activity of components of the Protein C anticoagulant pathwayof blood coagulation.

BACKGROUND OF THE INVENTION

Maintenance of proper hemostasis is the result of a careful balancebetween procoagulant and anticoagulant activities. After a trauma,coagulation is triggered primarily through activation of coagulationFactors IX and X (FIX, FX) by tissue factor (also denoted tissuethromboplastin) and Factor VII (FVII) followed by generation ofthrombin, which in turn cleaves fibrinogen to form soluble fibrin. Aftercrosslinking by Factor XIII, a three-dimensional insoluble gel clot isobtained which prevents further blood losses.

Regulation of this highly potent system, shown schematically in FIG. 1,is accomplished by a balanced relation between procoagulant activities(shown with solid line arrows) and anticoagulant activities (shown withdashed line arrows). The anticoagulant activities include (1) inhibitionof thrombin by antithrombin (AT) and α₂-macroglobulin, and (2)prevention of further thrombin formation by the Protein C anticoagulantpathway. In that pathway, activated Protein C (APC) inactivates thecoagulation proteins Factor VIII and Factor V in their activated forms(FVIIIa, FVa) through proteolytic cleavage. In addition, Factor Xa isinhibited by antithrombin and tissue factor pathway inhibitor (TFPI),the latter also inhibiting the tissue factor/Factor VIIa complex. FactorVIIIa and Factor Va have potent procoagulant activities as cofactors inthe activation of Factor X and prothrombin, respectively, and increasethe reaction rates of these processes about 1000-fold each. Therefore,the inactivation of Factors Va and VIIIa by APC essentially stopsfurther thrombin generation, thus providing a strong anticoagulanteffect. Protein S and Factor V act as cofactors to activated Protein C(APC).

As shown in FIG. 1, the activation of coagulation through the intrinsicor extrinsic systems results in the activation of Factor X, a keycomponent in the final common pathway. In the intrinsic system, theinitial event is the activation of contact factors (Factor XII,prekallikrein) followed by the activation of Factor XI, which in turnactivates Factor IX. In the extrinsic system, Factor IX and Factor X areactivated by the tissue factor/Factor VIIa complex.

As FIG. 1 shows, calcium ions have to be present in several of thesereactions. The activation of Factor X by Factor IXa, and of prothrombinby Factor Xa, also requires procoagulant phospholipids. In vivo, this isprovided by the membrane surface of activated platelets; in vitro byplatelet extracts, purified phospholipids, synthetic phospholipidsand/or crude phospholipid extracts from suitable sources. The total andfree calcium ion concentrations in native plasma are about 2.4 and 1.2mmol/L, respectively. Typically, calcium ion concentrations used inanalytical methods for the determination of coagulation oranticoagulation factors are in the range 1.5-10 mmol/L. Theconcentrations of other metal ions in plasma are lower, with typicalvalues for the total concentration being 1 mmol/L for Mg²⁺ and 5-40μmol/L for Zn²⁺, Cu²⁺ and Mn²⁺.

Defects in the Protein C anticoagulant pathway may increase the risk ofthrombosis due to a decreased capacity to prevent thrombin formation.Such defects may be due to deficiencies in the activity of Protein Cand/or its cofactor Protein S. Another recently detected defect is apoint mutation in the Factor V gene (G→A) at nucleotide 1691, resultingin the amino acid substitution Arg (R)→Gln (Q) at position 506 in FactorV/Factor Va, denoted FV:Q⁵⁰⁶ or Factor V Leiden. Heterozygosity andhomozygosity for this mutation are often denoted FV:R506Q and FV:Q506Q,respectively. This mutation is at one of the three APC cleavage sites(amino acids 306, 506, 679) in Factor Va, impairs its degradation byactivated Protein C (APC), and confers a condition denoted as APCresistance.

APC resistance is to be considered a blood coagulation disorderrecognized by an abnormally low anticoagulant response to activatedProtein C (APC), and the determination of APC resistance may be used toscreen for and diagnose thromboembolic diseases, such as hereditarythrombophilia, or for determining the risk for a human to acquire amanifestation of this blood coagulation disorder (e.g., European Pat.No. 608235).

Hence, there is a need to investigate these components of the Protein Canticoagulant pathway in the evaluation of thrombotic patients, andpotentially also to screen for abnormalities of Protein C, Protein S andFactor V anticoagulant activity in situations connected with anincreased risk of thrombosis, such as before surgery, during and aftertrauma, during pregnancy, or in connection with the use of oralcontraceptive pills or hormone replacement therapy. Currently, clottingand/or chromogenic assays are available for analysis of Protein C andProtein S activity as well as for the detection of APC resistance (atleast 90% of which is due to the FV:Q⁵⁰⁶ mutation).

Protein C activity is typically measured after activation of endogenousProtein C, contained in a plasma sample, by thrombin or by a snake venomenzyme from Agkistrodon contortrix contortrix (e.g., European Patent203509 to Stocker), commercially available as the reagent Protac®C(Pentapharm AG, Basel, Switzerland). The concentration of Protac®C inthe activation mixture is typically about 0.1 U/mL or higher sinceotherwise an insufficient activation of Protein C may be obtained(Martinoli et al. (1986), Thromb. Res. 43:253-264; McCall et al. (1987),Thromb. Res. 45:681-685).

After activation by Protac®C, the protein C activity is determined witha clotting or chromogenic assay (Bertina (1990), Res. Clin. Lab.20:127-138; Marlar et al. (1989), Hum. Pathol. 20:1040-1047; EuropeanPat. No. 486515). In clotting methods, coagulation is triggered throughthe intrinsic pathway by using APTT reagents or through the extrinsicpathway with the use of tissue factor. In both cases calcium ions areadded to a final concentration of usually 5-10 mmol/L. Commercial kitsand reagents are available for the determination of Protein C activity,such as Acticlot™ C (American Diagnostica GmbH, Pfungstadt, Germany),Stachrom Protein C (Diagnostica Stago, Asnières, France), StaclotProtein C (Diagnostica Stago, Asnières, France), Coamatic® Protein C(Chromogenix AB, Mölndal, Sweden) and Protein C Activator (Dade Behring,Deerfield, Ill.).

The activation of Protein C by thrombin is stimulated about 1000-fold bythrombomodulin, an endothelial cell membrane protein (Esmon et al.(1981), Proc. Natl. Acad. Sci. (USA) 78:2249-2252). The use ofthrombin/thrombomodulin as activator of Protein C for analysis ofProtein C and/or Protein S activity in plasma samples, utilizing aphotometric method, is also known (French Pat. Appln. No. 2689 640-A1).

Protein S activity is determined from its stimulation of the activity ofAPC in its degradation of Factor Va and/or Factor VIIIa. Typically, insuch assays a standardized amount of APC is added to a plasma sample oractivation of endogenous protein C is performed whereafter the clottingtime is determined after a simultaneous or separate coagulationactivation via the intrinsic system using an APTT reagent, via theextrinsic system using tissue factor or Factor Xa (Bertina (1990),supra; Preda et al. (1990), Thromb. Res. 60:19-32; D'Angelo et al.(1995), Thromb. Res. 77:375-378). Chromogenic activity assays forprotein S have also been published, utilizing Factor IXa as an activatorand monitoring Factor Xa generation (European Pat. No. 567 636) orthrombin generation (European Pat. No. 486 515). In all these methods,calcium ions are added as mentioned above.

The FV:Q⁵⁰⁶ mutation in the Factor V molecule may be detected withmolecular biology methods based upon the use of the polymerase chainreaction (PCR) technique (Bertina et al. (1994), Nature 369:64-67), orby methods in which the functional activity of APC is determined. Suchfunctional activity methods may be coagulation-based (e.g., EuropeanPat. No. 608235; Rosèn et al. (1994), Thromb. Haemost. 72:255-260), andmay include the use of predilution of sample plasma with a plasma withlittle or no Factor V activity (European Pat. Appln. No. EP-A-94 905908.3; Jorquera et al. (1994), Lancet 344:1162-1163; Svensson et al.(1997), Thromb. Haemost. 77:332-335). The latter assay principle, acoagulation-based assay using predilution of sample plasma, is alsoutilized in a commercial product, Coatest® APC Resistance V (ChromogenixAB). Alternatively, chromogenic methods may be used (European Pat. No.608 235; Rosèn et al. (1995), Thromb. Haemost. 73:1364, Abstract 1778;Nicolaes et al. (1996), Thromb. Haemost. 76:404-410).

Since genetic defects in the Protein C anticoagulant pathway are foundin about 25% of unselected patients with venous thromboembolism (VTE)and in about 50% of patients with thrombophilia (i.e., patients fromfamilies with an increased tendency to VTE), there is a need for asingle test which detects all such abnormalities with a high sensitivityand specificity, i.e., a global (overall) test. One concept for a globaltest is based upon the activation of Protein C in plasma with Protac®Cand activation of coagulation via the intrinsic or extrinsic pathway(U.S. Pat. No. 5,001,069; European Pat. Appln. No. 696 642). Resultsobtained with a commercial kit application of this test, ProC Global(Behring Diagnostica, Marburg, Germany), in which intrinsic activationof coagulation is accomplished through addition of an APTT reagent, showa sensitivity for Protein C deficiency, Protein S deficiency, and theFV:Q⁵⁰⁶ mutation of, respectively, about 90%, 50-80% and more than 90%on analysis of healthy individuals and thrombotic patients (Dati et al.(1997), Clin. Chem. 43:1719-1723; Ruzicka et al. (1997), Thromb. Res.87:501-510). The specificity, however, is about 50% in thromboticcohorts and, therefore, a substantial proportion of positive results areobtained which can not be linked to known defects in components of theProtein C anticoagulant pathway, such as in protein C, protein S andFactor V. Thus, this test lacks sufficient specificity.

Furthermore, results from analysis of pregnant women lacking any of theknown defects in the Protein C anticoagulant pathway are clearlydifferent from analysis of normal healthy individuals (Rang{dot over(a)}rd et al. (1997), Annals Hematol. 74, Supplement II, Abstract 74,A77; Siegemund et al. (1997), Annals Hematol. 74, Supplement II,Abstract 188, A105), which necessitates separate ranges for thesecohorts. This as yet uncharacterized interference limits the generalapplicability of the test. An alternative global method for thedetection of defects in the protein C anticoagulant pathway, based uponactivation of endogenous plasma Protein C by Protac®C utilizes tissuefactor as the trigger of the coagulation (Preda et al. (1996), BloodCoag. Fibrinol. 7:465-469). The sensitivity of this method also islimited, especially for Protein S. Furthermore, different samplecategories, e.g., pregnant and non-pregnant women, may require differentapproaches for evaluation of the results due to interference fromfactors not related to known defects of the Protein C anticoagulantpathway.

For a global test to be useful as a screening test for known inheriteddefects in the Protein C anticoagulant pathway (e.g., Protein Cdeficiency, protein S deficiency or the FV:Q⁵⁰⁶ mutation), thesensitivity should be high, at least 90%, for all these defects.Furthermore the specificity should be high, above 60%, preferably above70%, and more preferably above 80%, in order to considerably reduce thenumber of false positive results. The state-of-the-art methods do notprovide a satisfactory solution to these requirements. For thedevelopment of improved methods for the specific determination ofProtein C and Protein S activity, and for determination of mutations inFactor V which affect its anticoagulant activity, it is also desirableto improve the resolution and specificity of these methods. There isalso a need to improve the stability of different reagents used in suchmethods

Thus, the technical problem underlying the present invention is theprovision of in vitro methods with improved sensitivity and specificityfor diagnostic screening and for the specific detection of defects inthe Protein C anticoagulant pathway in humans. A further recognizedproblem is to improve the stability of reagents used in such methods.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the surprising findingthat the addition of low levels of divalent metal ions, such as Mg²⁺,Mn²⁺, Zn²⁺, Ni²⁺, Sr²⁺, or Cu²⁺ ions, or of the monovalent copper ion(Cu⁺), in the presence of calcium ions, enhances the anticoagulantactivity of the Protein C anticoagulant pathway, provides for a highdegree of resolution between different levels of Protein C activity andProtein S activity, provides a high degree of discrimination for thepresence of the FV:Q⁵⁰⁶ mutation, and results in an improved sensitivityand specificity for detection of defects in components of the Protein Canticoagulant pathway with photometric and/or clotting methods. Thus,the invention also constitutes a “global” method for detecting defectsin the Protein C anticoagulant pathway. In addition, the addition ofdivalent metal ions provides for an unexpected improvement of thestability of the reagents typically used in determining theanticoagulant activity of components of the Protein C anticoagulantpathway.

Thus, in one aspect, the present invention provides an in vitro methodfor qualitative screening and quantitative determination of thefunctional activity of components of the Protein C anticoagulant pathwayof blood coagulation, comprising measuring the conversion rate of anexogenous substrate by an enzyme, the activity of which is related toProtein C anticoagulant activity, in a human blood or plasma samplecomprising coagulation factors and said exogenous substrate, after atleast partial activation of coagulation through the intrinsic, extrinsicor common pathways and triggering coagulation by adding calcium ions;and comparing said conversion rate with the conversion rate of a normalhuman blood or plasma sample determined in the same way, said methodbeing characterized by adding further metal(s) ions to said sample.

The present invention is thus concerned with in vitro methods forscreening for, in a human, defects in the Protein C anticoagulantpathway due to, for example, Protein C deficiency, Protein S deficiency,or Factor V mutations such as the FV:Q⁵⁰⁶ mutation, or other Factor Vdefects related to APC resistance and/or APC cofactor activity. Suchmethods may be designed for the specific detection of Protein Cdeficiency, Protein S deficiency, or mutations in Factor V/Factor Vawhich affect the cleavage rate by APC. One preferred embodiment of thepresent invention comprises a global test for the Protein Canticoagulant pathway.

The methods of the invention allow improved screening and diagnosis ofdefects in the Protein C anticoagulant pathway in patients withthromboembolic diseases such as deep venous thrombosis and/or pulmonaryembolism. In cases where a patient belongs to a family with hereditarythrombophilia, the methods are also suitable for the investigation offamily members of the patient to determine the possible inheritance ofdefects within the pathway. The methods are also particularly useful fordiagnosing defects in the Protein C anticoagulant pathway in patientsbefore surgery, patients with trauma, in pregnant women, or in womenreceiving oral contraceptive pills or hormone replacement therapy suchas estrogen therapy. Furthermore, the “global” methods of the inventionmay be used for the detection not only of known defects in the Protein Canticoagulant pathway, but also of hitherto unrecognized defects. Inparticular, the invention provides for specific photometric and/orclotting methods for such unrecognized defects in the Protein Canticoagulant pathway.

The methods of the invention may comprise monitoring the conversion ofan exogenous photometric substrate for either Factor Xa or thrombin,containing a chromophore, fluorophore or luminophore as a leaving group.Examples of such photometrically measurable leaving groups arep-nitroaniline (a chromophore) for use in colorimetric methods;naphthylamine and coumarin derivatives such as methylcoumarine,aminoisophthalic acid and its derivatives (fluorophores) for use influorimetric methods; and isoluminolamide (a luminophore) for use inluminometric methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the blood coagulation system andits regulation.

FIG. 2 is a graphic representation of the results obtained in Example 2,i.e., the effects of the metal ions in a chromogenic Protein C assay.

FIG. 3 is a graphic representation of the results obtained in Example 5,i.e., the effect of Mg²⁺ and Mn²⁺ in a chromogenic Protein S assay.

FIG. 4 is a graphic representation the results obtained in Example 8,i.e., the effect of metal ions on discrimination for Protein Sdeficiency and for FV:Q⁵⁰⁶ in a global Protein C pathway assay usingFactor Xa as activator.

FIG. 5 is a graphic representation of the results obtained in Example12, i.e., the effect of Mg²⁺ in a global chromogenic assay for thedetection of Protein C deficiency, Protein S deficiency, and the FV:Q⁵⁰⁶mutation using a recombinant tissue factor.

FIG. 6 is a graphic representation of the results obtained in Example13, i.e., the effect of Mn²⁺ on the determination of free Protein Sactivity in a chromogenic thrombin generation assay.

FIG. 7 is a graphic representation of the results obtained in Example14, i.e., the effect of Mg²⁺ and Mn²⁺ on the determination of Protein Cactivity in a chromogenic thrombin generation assay.

FIG. 8 is a graphic representation of the results obtained in Example15, i.e., the effect of Mg²⁺ and Mn²⁺ on the detection of Protein Cdeficiency, Protein S deficiency and the FV:Q⁵⁰⁶ mutation in a globalchromogenic method using a recombinant tissue factor as activator ofcoagulation and monitoring thrombin generation.

DETAILED DESCRIPTION OF THE INVENTION

Definitions.

In order to more clearly and concisely describe and point out thesubject matter of the claimed invention, the following definitions areprovided for specific terms which are used in the following writtendescription and the appended claims.

As used herein, the term “additional metal ions” refers to divalentmetal ions, such as Mg²⁺, Mn²⁺, Zn²⁺, Ni²⁺, Sr²⁺, or Cu²⁺, to monovalentcopper ions (Cu⁺), or to mixtures thereof. These additional metal ionsmay be used in the methods and products of the invention in addition tothe Ca²⁺ ions which are typically used in such methods and products.

As used herein, the term “blood sample” is defined to cover a bloodsample, such as whole blood, or a blood derived sample, such as a bloodplasma sample or a blood serum sample.

As used herein, the term “photometric assay” is defined to includecolorimetric, fluorimetric and luminometric assay methods.

As used herein, the term “coagulation factors” refers to the factors ofthe blood coagulation pathway comprising components in the intrinsic,extrinsic and common coagulation pathways (procoagulant events see FIG.1), or in the Protein C anticoagulant pathway (anticoagulant events seeFIG. 1). The term embraces such factors whether they are present in asample as endogenous components (i.e., being inherent in the bloodsample), or whether they have been added as exogenous factors.Phospholipid(s) may also be included as coagulation factors when addedin a method utilizing any of the intrinsic, extrinsic or common pathwaysfor activation of coagulation.

The present invention is based, in part, upon the surprising findingthat the addition of low levels of divalent metal ions, such as Mg²⁺,Mn²⁺, Zn²⁺, Ni²⁺, Sr²⁺, or Cu²⁺ ions, or of the monovalent copper ion(Cu⁺), in the presence of calcium ions, enhances the anticoagulantactivity of the Protein C anticoagulant pathway, provides for a highdegree of resolution between different levels of Protein C activity andProtein S activity, provides a high degree of discrimination for thepresence of the FV:Q⁵⁰⁶ mutation, and results in an improved sensitivityand specificity for detection of defects in components of the Protein Canticoagulant pathway with photometric and/or clotting methods.

The present invention is most unexpected in light of the prior art,which teaches that several divalent ions increase the procoagulantactivity of certain vitamin K-dependent coagulation factors in thepresence of calcium ions (see procoagulant events in FIG. 1). Therefore,it would not be expected that the addition of such metal ions couldimprove tests relating to anticoagulant activity, and specifically tothe Protein C anticoagulant pathway.

Thus, for example, it was known that Mg²⁺ stimulates the activity ofFactor IXa and also enhances the activation rate of Factor IX by FactorXIa and tissue factor (Byrne et al. (1980), J. Biol. Chem.255:1430-1435; Sekiya et al. (1995), J. Biol. Chem. 270:14325-14331;Sekiya et al. (1996), J. Biol. Chem. 271:8541-8544; Morita et al.(1997), Thromb. Haemost. 78, Supplement 430, Abstract PS-1755). It wasalso shown that Protein C, prothrombin, Factor VII and Factor X are notresponsive to Mg²⁺ (Sekiya et al. (1995), supra). Mg²⁺ also has beenshown to stimulate prothrombin activation by Factor Xa, phospholipid andcalcium ions (Prendergast et al. (1997), J. Biol. Chem. 252: 840-850),an effect which, however, may not be pronounced at calcium ionconcentrations above 1 mmol/L (Sekiya et al. (1995), supra).Furthermore, it has been shown that Factor IX has a unique binding sitefor Mn²⁺(Amphlett et al. (1978), J. Biol. Chem. 253:6774-76779), whichsite has been suggested to be identical with the Mg²⁺ binding site(Sekiya et al. (1995), supra). Mn²⁺ ions have also been shown to enhancethe binding of Factor IX to procoagulant phospholipids in the presenceof calcium or Sr²⁺ ions, the latter thus also having a procoagulanteffect (Liebman et al. (1987), J. Biol. Chem. 262:7605-7612).

Furthermore, Mg²⁺ and Mn²⁺ ions have been shown to increase theamidolytic activity of Factor VIIa, i.e., the cleavage rate of lowmolecular weight synthetic peptide substrates (Butenas et al. (1994),Biochemistry 33:3449-3456; Persson et al. (1995), Eur. J. Biochem.234:293-300), whereas Zn²⁺ ions have been reported to have an inhibitoryeffect on the amidolytic activity of Factor VIIa but no effect on theamidolytic activity of Factor Xa, thrombin or activated Protein C(Pedersen et al. (1991), Thromb. Haemost. 65:528-534). Mn²⁺ions havealso been shown to substitute for calcium ions in the activation ofFactor X by Russell's Viper Venom enzyme, albeit providing a loweractivation rate (Bajaj et al. (1977), J. Biol. Chem. 252:4758-4761).

The prior art also teaches that divalent metal ions such as Zn²⁺ andCu²⁺ stimulate the autoactivation of Factor XII, a non-vitaminK-dependent coagulation factor (Shore et al. (1987), Biochemistry26:2250-2258; Bernardo et al. (1993), J. Biol. Chem. 268:12468-12476).

In addition, the prior art teaches that Mg²⁺ and Mn²⁺ stimulate theinhibition of APC by the two plasma protease inhibitors α₂-macroglobulinand plasmin inhibitor (Heeb et al. (1991), J. Biol. Chem.266:17606-17612). Thus, the addition of Mg²⁺ and Mn²⁺ under theconditions used results in a decreased anticoagulant activity of APC.

Therefore, the present invention, which provides an increasedanticoagulant activity of the Protein C anticoagulant pathway, providesfor a high degree of resolution between different levels of Protein Cactivity and Protein S activity, provides a high degree ofdiscrimination for the presence of the FV:Q⁵⁰⁶ mutation, and results inan improved sensitivity and specificity for detection of defects in theProtein C anticoagulant pathway, through the use of metal ions inaddition to calcium ions, could not be derived or expected from theprior art knowledge in the field.

A preferred embodiment of the present invention thus covers a method forthe global screening for defects in the Protein C anticoagulant pathwayof blood coagulation in a human, comprising

(a) incubating a blood sample of said human comprising coagulationfactors with:

(1) an activator for the Protein C in the sample,

(2) a suitable coagulation activator,

(3) an exogenous synthetic substrate for either Factor Xa or thrombincomprising a photometrically measurable leaving group,

(4) calcium ions, and

(5) additional metal ions;

(b) determining the conversion rate of said exogenous substrate; and

(c) comparing said conversion rate with the conversion rate of a normalhuman blood sample determined in the same way.

A further preferred embodiment of the present invention relates to amethod for the determination of free Protein S activity, comprising:

(a) incubating a human blood sample comprising coagulation factors with:

(1) exogenous activated Protein C or exogenous Protein C together withan activator of Protein C,

(2) a suitable coagulation activator,

(3) an exogenous synthetic substrate for either Factor Xa or thrombincomprising a photometrically measurable leaving group,

(4) calcium ions, and

(5) additional metal ions;

(b) determining the conversion rate of said exogenous substrate; and

(c) comparing said conversion rate with the conversion rate of a normalhuman blood sample determined in the same way.

Another preferred embodiment of the present invention relates to amethod for the determination of Protein C activity, comprising:

(a) incubating a human blood sample comprising coagulation factors with:

(1) an activator for the Protein C in said sample,

(2) a suitable coagulation activator,

(3) an exogenous synthetic substrate for either Factor Xa or thrombincomprising a photometrically measurable leaving group,

(4) calcium ions, and

(5) additional metal ions;

(b) determining the conversion rate of said exogenous substrate; and

(c) comparing said conversion rate with the conversion rate of normalhuman blood sample determined in the same way.

A fourth preferred embodiment of the present invention is a method forscreening for Factor V mutation(s) in a human blood sample, comprising:

(a) incubating a human blood sample comprising coagulation factors with:

(1) exogenous activated Protein C, or exogenous Protein C together withan activator of Protein C, or an activator for endogenous Protein C,

(2) a suitable coagulation activator,

(3) an exogenous synthetic substrate for either Factor Xa or thrombincomprising a photometrically measurable leaving group,

(4) calcium ions, and

(5) additional metal ions;

(b) determining the conversion rate of said exogenous substrate; and

(c) comparing said conversion rate with the conversion rate of a normalhuman blood sample determined in the same way.

In the above preferred methods for global screening for defects in theProtein C anticoagulant pathway, for the determination of free Protein Sactivity or Protein C activity, or for screening for Factor Vmutation(s) such as the FV:Q⁵⁰⁶ mutation in a blood sample, step (a)comprises incubating the blood sample comprising coagulation factors inthe presence of the additional metal ions used according to the presentinvention with

(1) an activator for the Protein C in said sample to provide activationof endogenous Protein C, or exogenous activated Protein C or exogenousProtein C together with an activator of Protein C,

(2) a suitable coagulation activator to provide at least partialactivation of coagulation,

(3) an exogenous synthetic substrate for either Factor Xa or thrombincomprising a photometrically measurable leaving group to provide formonitoring Factor X_(a) or thrombin activity and

(4) calcium ions to trigger coagulation.

These steps (1) to (4) can be performed simultaneously, separatelyand/or in different sequential combinations, providing for “one-step” to“four-step” methods as follows:

In a one-step method, all components necessary for performing steps (1)to (4) above are added simultaneously.

In two-step methods, (a) the components for steps (1) and (2) may firstbe combined followed by simultaneous addition of the exogenous substratewith calcium ions (3) and (4); or (b) all the components except thecalcium ions may be added simultaneously, the addition of calcium ionsthen comprising the second step; or (c) the components for steps (1),(2) and (4) may be included simultaneously and step (3) be performed asa separate step.

In three-step methods, (a) steps (1) and (2) are combined and steps (3)and (4) are performed as separate steps; or (b) steps (1) and (2) areperformed as separate steps and steps (3) and (4) are performedsimultaneously; or (c) steps (2) and (3) are performed as separatesteps, and steps (I) and (4) are performed simultaneously; or (d) step(1) is performed as a separate step, steps (2) and (4) are performedsimultaneously, followed by step (3).

In four-step methods steps (1) to (4) are performed as separate steps inthe order as described or in any other different order.

All one-, two-, three- or four-step methods may be used in chromogenic,fluorimetric and luminometric methods. Alternatively, in otherembodiments of the invention, the methods may comprise measurements ofclotting times, utilizing activation through the intrinsic, extrinsic orcommon pathways.

For any method, the additional metal ions used according to the presentinvention may be added either initially or at a later stage to any ofthe reagents. In applications where endogenous Protein C is activated bya Protein C activator, such as in methods for Protein C activity andglobal methods for the Protein C anticoagulant pathway, one preferredmode of the invention is to include the additional metal ions in theProtein C activation step, for example when Protac®C is used as theProtein C activator.

Specifically, the invention concerns the addition of divalent metal ionsor of the monovalent copper ion, in order to increase the resolutionbetween different levels of Protein C activity or Protein S activity, aswell as to provide a high degree of discrimination for the presence ofthe FV:Q⁵⁰⁶ mutation, resulting in an improved sensitivity andspecificity for detection of defects in the Protein C anticoagulantpathway of blood coagulation. The range of concentrations of metal ionswithin which the anticoagulant activity of the Protein C system isstimulated varies with respect to the particular metal ion. Theconcentration range for metal ions other than Mg²⁺ is preferably 1μmol/L-2 mmol/L, more preferably 5-400 μmol/L, and most preferably 10-80μmol/L. For Mg²⁺ ions, the concentration range is preferably 20μmol/L-10 mmol/L, more preferably 100 μmol/L-2 mmol/L, and mostpreferably 200 μmol/L-1 mmol/L.

The metal ions of the invention will typically be provided incombination with negatively charged counter ions. The counter ionsshould be selected to allow the metal ions to be available in solutionat the above described concentrations. Suitable counter ions are mono-,di- and trivalent anions, preferably mono- and divalent anions, such aschloride, sulfate and nitrate anions. Alternatively, the metal ionscould be provided in the form of metal ion complexes with proteins suchas blood proteins, or on a solid surface such as a metal ion coated wallof a reaction vessel.

In preferred embodiments of the invention, photometric methods are usedfor monitoring the anticoagulant activity of components in the Protein Canticoagulant pathway. In such photometric methods, synthetic substrateswith colorimetric, fluorimetric or luminometric leaving groups are used,which substrates preferably should be selective for Factor Xa or forthrombin. Because the generation of Factor Xa and thrombin is influencedby the Protein C anticoagulant activity in the reaction mixture, themeasurement of the conversion rate of such substrates may be used forthe determination of the activity of the Protein C anticoagulantpathway. The measurement of the conversion rate may be compared with thecorresponding conversion rate obtained when using a normal human bloodsample, or human pooled normal pool, as a test sample. The conversionrate may be measured kinetically (e.g., by monitoring the change inoptical density (OD) versus time, expressed as e.g. ΔOD/min), ormeasured after a fixed incubation time, expressed as OD.

The determination of the conversion rate of synthetic substrates isperformed with instruments suitable for monitoring the release of theleaving group from the particular substrate chosen. When the conversionrate is determined in a microplate reader, it is preferred to performreadings in the “dual wavelength mode” in order to eliminate possibledifferences between microplate wells. In this mode, one wavelength isselected for detecting the release of the leaving group, and anotherwavelength is selected in a range where the leaving group does not haveany appreciable absorbance. When a calorimetric leaving group such asparanitroaniline (pNA) is chosen, a suitable dual wavelength reading maybe performed at 405 and 490 nanometers, expressed as OD_(405-490 nm).

In preferred photometric methods, diluted blood samples are used inorder to avoid interference of blood sample components in the testsample. The concentration of the blood sample in the final sample (i.e.,the sample for which the optical density is determined) may varydepending on the actual method used. For example, for colorimetricmethods the blood sample concentration may be below 10%, and ispreferably below 5%.

The methods of the present invention allow for convenient reaction timessuch as 1-10 min, preferably 2-5 min, to provide for easy applicabilityto automated coagulation instruments.

A variety of activators of Protein C may be used in the methods of thepresent invention. For example, thrombin may be employed with or withoutthrombomodulin, and may be obtained from human or non-human sources, ormay be produced by recombinant technology. Recombinantly producedprotein may have either the wild-type protein sequence or a modifiedprotein sequence which still provides suitable functional activity.Alternatively, a snake venom enzyme which activates Protein C may beemployed. Suitable snake venom enzymes are preferably obtained from orderived from the Agkistrodon snake family, and may be added as crudevenom, or in a purified preparation such as the product Protac®C.Suitable snake venom enzymes may also be produced by recombinanttechnology, having either the wild-type protein sequence or a modifiedprotein sequence which still provides suitable functional activity. Theconcentration of the Protein C activator will vary depending on theparticular assay conditions used. Thus, for the Protac®C activator, theconcentration may vary between 1×10⁻³ and 1 U/mL, preferably 2×10⁻³ and0.3 U/mL during the activation of Protein C in a global method fordetermining activity of the Protein C anticoagulant pathway, or inspecific methods for determining Protein C and/or free Protein Sactivity, or for methods for detection of mutations in Factor V/FactorVa which affect the cleavage rate by APC.

In preferred embodiments of the present invention, the activation ofProtein C in the blood sample precedes or occurs simultaneously with theactivation of coagulation.

In preferred methods of the present invention, a suitable activator ofcoagulation is added to the assay medium or sample. Such activators maybe selected to activate the intrinsic, extrinsic or common pathways ofthe coagulation cascade.

For example, activation through the intrinsic system may be accomplishedwith an APTT reagent containing a suitable contact activator, or withthe separate addition of a contact activator. Suitable activators of theintrinsic pathway include compositions of phospholipids and contactactivators. As contact activators, ellagic acid, collagen or collagenrelated substances, or various forms of silica (kaolin, micronizedsilica, colloidal silica) may be used. Alternatively, rather than usinga contact activator, Factor XIIa, Factor XIa or Factor IXa may be usedin combination with phospholipids as activators of the intrinsicpathway. Optionally, components such as prothrombin, Factor VIII/FactorVIIIa and Factor X may be added. Photometric substrates selective forFactor Xa or thrombin may also be used.

Activation through the extrinsic system may be accomplished by theaddition of tissue factor, with or without the addition of FactorVIII/Factor VIIa. The tissue factor may be obtained or derived fromhuman or non-human sources, or may be produced by recombinanttechnology. Recombinantly produced protein may have either the wild-typeprotein sequence or a modified protein sequence which still providessuitable functional activity. Alternatively, activation may beaccomplished by Factor VIIa in combination with phospholipids.Optionally, reagents such as prothrombin, Factor V/Va, Factor IX andFactor X may be added. Photometric substrates selective for FXa orthrombin may also be used.

Activation of the common pathway may be accomplished by addition ofexogenous Factor Xa, or by addition of exogenous Factor X in combinationwith an exogenous activator of Factor X, such as a snake venom enzyme(e.g., snake venom enzyme from Russelli Viperii). Alternatively, theexogenous activator of Factor X may be added for activation ofendogenous Factor X. Optionally, prothrombin and/or Factor V/Va may beadded. Photometric substrates selective for thrombin may also be used.

In the above described modes for the activation or partial activation ofcoagulation according to the intrinsic, extrinsic or common pathways,phospholipids may be added as a mixture of synthetic phospholipidsand/or purified phospholipids, or as crude extracts from biologicalsources such as brain, platelets, placenta, egg yolks or soybeans.

Generally, interference due to variable functional levels of componentsin the sample may be minimized by adding to the assay medium or sample asufficient amount of plasma deficient in the Protein C anticoagulantpathway component to be measured. Thus, when measuring Protein C,Protein S, or Factor V activity, plasmas deficient in Protein C, ProteinS or Factor V, respectively, may be employed. In the case of a globalmethod for the Protein C anticoagulant pathway, a plasma deficient ineach of Protein C, Protein S and Factor V may be added.

Thus, for example, in the case of a method for determining Protein Sactivity or a method for detection of a Factor V mutation which affectsthe degradation of Factor V/Va by APC, exogenous Protein C may be addedin the form of a plasma deficient in Protein S or Factor V,respectively. Similarly, Protein S may be added in methods for Protein Cactivity or for the detection of mutations in Factor V. In the case ofmethods for determining Protein C and Protein S activity, FactorV/Factor Va may also be added to minimize interference from mutatedFactor V which may be present in the sample.

The above-mentioned coagulation factors and components of the Protein Canticoagulant pathway, namely Factor XIIa, Factor XIa, Factor IX/IXa,Factor VIII/VIIIa, Factor VII/VIIa, Factor X/Xa, Factor V/Va,prothrombin, Protein C/APC and Protein S, may be of human or non-humanorigin, but are preferably of bovine or human origin. Said coagulationfactors may also be produced by recombinant technology and havewild-type protein sequences or modified protein sequences which stillprovide suitable functional activity.

In preferred embodiments, in order to prevent fibrin gel formation, afibrin polymerization inhibitor may be added to the reaction mixture,such as Gly-Pro-Arg-Pro or others known in the art.

In chromogenic methods, any chromogenic substrates for Factor Xa may beused, such as Benzoyl-Ile-Glu-Gly-Arg-pNA (S-2222, Chromogenix AB),N-a-Z-D-Arg-Gly-Arg-pNA (S-2765, Chromogenix AB),CH³SO₂-D-Leu-Gly-Arg-pNA (CBS 31.39, Diagnostica Stago, Asniéres,France) and MeO-CO-D-CHG-Gly-Arg-pNA (Spectrozyme Xa, AmericanDiagnostica, Greenwich, USA). Correspondingly, any chromogenicsubstrates for thrombin may be used, such as H-D-Phe-Pip-Arg-pNA(S-2238, Chromogenix AB), pyroGlu-Pro-Arg-PNA (S-2366, Chromogenix AB),H-D-Ala-Pro-Arg-pNA (S-2846, Chromogenix AB), Z-D-Arg-Sarc-Arg-pNA(S-2796, Chromogenix AB), AcOH*H-D-CHG-But-Arg-pNA (CBS 34.47,Diagnostica Stago) and H-D-HHT-Ala-Arg-pNA (Spectrozyme TH, AmericanDiagnostica).

In another aspect, the present invention provides kits for use in theabove-described methods. In one preferred embodiment, a kit is providedcomprising the following components:

(a) an activator for the endogenous Protein C in a sample, or exogenousactivated Protein C, or exogenous Protein C together with an activatorof Protein C;

(b) a suitable coagulation activator;

(c) an exogenous synthetic substrate for either Factor Xa or thrombincomprising a photometrically measurable leaving group;

(d) calcium ions; and

(e) additional metal ions;

in separate containers and/or in containers comprising mixtures of atleast two of said components, and in aqueous solution or in lyophilizedform.

In another preferred embodiment, a kit is provided comprising thefollowing components:

(a) an activator for the endogenous Protein C in a sample, or exogenousactivated Protein C, or exogenous Protein C together with an activatorof Protein C;

(b) a suitable coagulation activator;

(c) an exogenous synthetic substrate for either Factor Xa or thrombincomprising a photometrically measurable leaving group;

(d) calcium ions;

(e) additional metal ions;

(f) coagulation factors; and

in separate containers and/or in containers comprising mixtures of atleast two of said components, and in aqueous solution or in lyophilizedform.

In another aspect, the present invention provides a reagent for use inthe above methods, and comprising said additional metal ions and atleast one, and preferably at least two, of the following components (a)to (e):

(a) an activator for the endogenous Protein C in the sample, orexogenous activated Protein C, or exogenous Protein C together with anactivator of Protein C;

(b) a suitable coagulation activator;

(c) an exogenous synthetic substrate for either Factor Xa or thrombincomprising a photometrically measurable leaving group;

(d) calcium ions; and

(e) one or more coagulation factors;

in one container in aqueous solution or in lyophilized form.

Thus, for example, in one preferred embodiment the reagent comprisesactivated Protein C, with or without calcium ions, as well as theadditional metal ions. In another preferred embodiment, the reagentcomprises coagulation factors and Protein C activators and, if desired,in combination with phospholipid(s), as well as the additional metalions. In other embodiments, the reagent comprises Factor IX/IXa, FactorX/Xa and/or calcium ions, and the additional metal ions, or may compriseFactor V/Va, Protein C and prothrombin in combination with theadditional metal ions. In another embodiment, Factor VIII/VIIIa and/orthrombin also may be combined with the additional metal ions. In yetanother embodiment, the additional metal ions may be combined with aProtein C activator, such as Protac®C or thrombin/thrombomodulin. In allcases, the reagents may be suitably provided in a container, in aqueoussolution or in lyophilized form.

A wide range of concentrations of reactants can be used in the methodsof the present invention. Table 1 below presents suitable and preferredranges for the various components used in the methods, and/or containedin the kits and reagents, as well as preferred ranges of pH and ionicstrength. Naturally, higher concentrations may be contained in the kits,allowing for dilution before use.

TABLE 1 Parameter Final concentration in final reaction medium Bloodsample 0.02-10%, preferably 0.1-5% (v/v) FIX/FIXa 1 × 10⁻¹⁵ − 1 × 10⁻⁶mol/L FX/FXa 1 × 10⁻¹⁵ − 5 × 10⁻⁷ mol/L FV/FVa 1 × 10⁻¹² − 10⁻⁷ mol/L,preferably 2 × 10⁻¹⁰ − 5 × 10⁻⁸ mol/L FVII/FVIIa 1 × 10⁻¹⁵ − 2 × 10⁻⁸mol/L FVIII/FVIIIa 1 × 10⁻⁴ − 5 × 10⁻¹ IU/mL Prothrombin 1 × 10⁻⁹ − 5 ×10⁻⁷ mol/L Thrombin 1 × 10⁻¹⁵ − 1 × 10⁻⁸ mol/L Ca²⁺ ions 0.5-20 mmol/L,preferably 1-10 mmol/L Mg²⁺ ions 20 μmol/L − 10 mmol/L, preferably 100μmol/L − 2 mmol/L, most preferably 200 μmol/L − 1 mmol/L. Mn²⁺, Zn²⁺, 1μmol/L − 2 mmol/L, preferably 5-400 μmol/L, Cu²⁺, Ni²⁺, Sr²⁺ mostpreferably 10-80 μmol/L and/or Cu⁺ ions Protein C/APC 1 × 10⁻¹⁰ − 1 ×10⁻⁷ mol/L, preferably 5 × 10⁻¹⁰ − 1 × 10⁻⁸ mol/L Protac ® C 1 × 10⁻³ −1 U/mL, preferably 2 × 10⁻³ − 0.3 U/mL Protein S 10⁻⁹ − 5 × 10⁻⁷ mol/LTissue factor 10⁻⁸ − 10⁻⁵ g/L, preferably 5 × 10⁻⁸ − 10⁻⁶ g/L Thrombin/10⁻¹¹ − 10⁻⁸ mol/L thrombomodulin Phospholipid 1 × 10⁻⁶ − 3 × 10⁻⁴ mol/LFibrin Range dependent on substance used polymerization inhibitorChromogenic 10⁻⁵ − 5 × 10⁻³ mol/L substrate pH 6.5-9.5, preferably 7-8.5Ionic strength 0-0.6, preferably 0.01-0.25

Suitable embodiments include preparations of one or more of thecomponents presented in Table 1, in aqueous solution or lyophilized in acontainer. For example, one or more of the proteins from Table 1 withand without additional metal ions, and optionally in the presence ofphospholipid(s). Such embodiments may comprise, for example, Protein Cor APC and said additional metal ions, optionally with calcium ions, andwith or without an active enzyme such as Factor IXa or Factor Xa. Otherembodiments may comprise the additional metal ions with one or more ofFactor V/Va, Protein S, prothrombin, Factor X, Factor VIII/VIIIa, andthrombin. A further embodiment may comprise a Protein C activator suchas Protac®C or thrombin/thrombomodulin with additional metal ions.

In particular, the present invention provides kits and reagents for usein the above-described in vitro methods for screening and diagnosing forProtein C and/or Protein S deficiency, and/or for mutations in Factor Vwhich affect the anticoagulant activity of APC. More generally, theinvention provides kits for in vitro methods for screening anddiagnosing for defects in the Protein C pathway, including but notlimited to those caused by Protein C deficiency, Protein S deficiency,and/or Factor V mutations.

EXAMPLES Example 1

The effect of manganese and magnesium ions on the determination ofProtein C activity in a three-stage, chromogenic, thrombin generationassay using the Protein C activator Protac®C was assessed as follows.

Samples: Protein C deficient plasma (Biopool AB, Ume{dot over (a)},Sweden) with and without addition of purified human Protein C(Chromogenix AB) to yield 0, 0.1, 0.5 and 1.0 IU/mL of Protein C.

Sample dilution: 1:41 in 25 mmol/L Tris-HCl, pH 7.6, 20 mmol/L NaCl,0.2% bovine serum albumin.

Protein C activator: Protac®C was used as a stock solution containing 10U/mL. Final concentration during activation of Protein C=0.17 U/mL. Mg²⁺and Mn²⁺ ions were added to yield final concentrations during activationof Protein C of 0.4 and 0.04 mmol/L, respectively.

Reagent 1:

Bovine Factor IXa (Enzyme Research, South Bend, Ill.), 180 pmol/L

Bovine FX (Chromogenix AB), 0.3 U/mL

Reagent 2:

Phospholipids*(Chromogenix AB), 60 μmol/L

Gly-Pro-Arg-Pro, 0.36 mg/mL (polymerization inhibitor)

Human Factor V, 0.2 U/mL

CaCl₂, 6 and 24 mmol/L (final conc. in assay=1.5 and 6 mmol/L)

A mixture of purified phospholipids containing 43% phosphatidylcholine,27% phosphatidylserine and 30% sphingomyelin.

Chromogenic thrombin substrate: S-2796 (Chromogenix AB), 2 mmol/L

Assay in a microplate: This assay is carried out as a three-stage methodcomprising, in the first stage, combining 50 μL of the diluted plasmawith 50 μL of the Protein C activator and incubating this mixture forthree minutes at 37° C., whereafter coagulation is activated by adding50 μL of Reagent 1 and 50 μL of Reagent 2 and incubating the mixture forfive minutes at 37° C., whereafter, in the third stage, the substratehydrolysis is carried out by adding 50 μL of the chromogenic thrombinsubstrate S-2796 and incubating for four minutes at 37° C. The reactionis then terminated by lowering the pH through addition of 50 μL of 20%acetic acid. Thereafter the optical density (OD) of the samples in themicrowells is recorded at 405 and 490 nm and the difference in opticaldensity between 405 and 490 nm, OD_(405-490 nm), is calculated. Thisthree-stage reaction is schematically shown as follows:

Protein C activation: Plasma dilution 50 μL Protein C activator 50 μL 3min, 37° C. Coagulation activation: Reagent 1 50 μL Reagent 2 50 μL 5min, 37° C. Substrate hydrolysis: S-2796 50 μL 4 min, 37° C. HOAc, 20%50 μL Recording of OD_(405-490 nm)

Results: The results are shown in the table below.

Protein C, IU/mL Ions 0 0.1 0.5 1.0 Ca²⁺, 6 mmol/L 0.542 0.515 0.5030.467 Ca²⁺, 1.5 mmol/L + Mn²⁺, 0.04 mmol/L 0.564 0.441 0.231 0.076 Ca²⁺,1.5 mmol/L + Mg²⁺, 0.4 mmol/L 0.541 0.441 0.230 0.069

The results demonstrate that by including manganese and magnesium ionsin a reaction system containing calcium ions, a strong enhancement ofthe anticoagulant activity is obtained, manifested by the fact thatincreasing concentrations of Protein C in the samples result in a muchdecreased absorbance, i.e. a much decreased thrombin generation. Incontrast, in the presence of calcium ions alone, there is a much lowerresolution in absorbance, i.e. in thrombin generation, at increasingProtein C concentrations. Thus, the addition of the additional metalions constitutes an improved method for determination of Protein Cactivity.

Example 2

The effect of different metal ions on the determination of Protein Cactivity in a three-stage thrombin generation assay using a four-foldlower concentration of Protein C activator was assessed.

Experimental conditions are as in Example 1, except for the use of afinal concentration of the Protein C activator (Protac®C) of 0.043 U/mL.Mg²⁺,Mn²⁺, Zn²⁺ and Ni²⁺ ions were added to yield final concentrationsduring activation of Protein C of 0.4 mmol/L (Mg²⁺) or 0.04 mmol/L. Zn²⁺ions, Mn²⁺ ions and Cu²⁺ ions were also added to yield a finalconcentration of 0.08 mmol/L. Ca²⁺ was also used at final concentrationsof 1.5 mmol/L and 6.6 mmol/L in the absence of other metal ions.

Results: The results are shown in the table below with all primary data,which also includes a comparison between final concentrations of 0.04and 0.08 mmol/L for Mn²⁺ and Zn²⁺.

Protein C, IU/mL Ions 0 0.1 0.5 1.0 Ca²⁺, 6 mmol/L 0.652 0.633 0.5590.505 Ca²⁺, 1.5 mmol/L 0.640 0.585 0.504 0.438 Ca²⁺, 1.5 mmol/L + Mn²⁺,0.04 mmol/L 0.725 0.689 0.503 0.237 Ca²⁺, 1.5 mmol/L + Mn²⁺, 0.08 mmol/L0.627 0.469 0.145 0.056 Ca²⁺, 1.5 mmol/L + Mg²⁺, 0.4 mmol/L 0.627 0.5830.361 0.123 Ca²⁺, 1.5 mmol/L + Zn²⁺, 0.04 mmol/L 0.513 0.446 0.334 0.129Ca²⁺, 1.5 mmol/L + Zn²⁺, 0.08 mmol/L 0.421 — 0.189 0.051 Ca²⁺, 1.5mmol/L + Ni²⁺, 0.04 mmol/L 0.594 0.437 0.173 0.047 Ca²⁺, 1.5 mmol/L +Cu²⁺, 0.08 mmol/L 0.487 0.425 0.348 0.099

These results, which are graphically shown in FIG. 2, demonstrate thatmany different metal ions provide an enhancement of the anticoagulantactivity, and also that a calcium concentration of 1.5 mmol/L in theabsence of any other additional metal ions lacks the anticoagulationenhancement property. Furthermore, the concentration of Protac®C is notcritical since the use of a four-fold lower concentration of thiscomponent still results in a pronounced anticoagulant activity in thepresence of additional metal ions.

Example 3

The effect of metal ions on the determination of Protein C activity in atwo-stage thrombin generation assay was assessed.

These experimental details are as described in Example 1, with thefollowing exceptions:

(a) the final concentration of the Protein C activator (Protac®C) was0.043 U/mL during activation of Protein C,

(b) the chromogenic thrombin substrate used was S-2846 (Chromogenix AB),

(c) the chromogenic substrate was included in Reagent 1,

(d) purified human Protein C was added to Protein C deficiency plasma toyield 0, 0.1 and 0.5 IU/mL, and

(e) the metal ions tested were Mn²⁺ and Mg²⁺.

Results: The results are shown in the table below expressed asOD_(405-490 nm):

Protein C, IU/mL Mg²⁺ ions 0 0.1 0.5 Ca²⁺, 6 mmol/L 1.18 1.07 0.669Ca²⁺, 1.5 mmol/L + Mn²⁺, 0.04 mmol/L 1.36 1.17 0.258 Ca²⁺, 6 mmol/L 1.241.05 0.554 Ca²⁺, 1.5 mmol/L + Mg²⁺, 0.4 mmol/L 1.45 1.15 0.215

These results show that a significantly higher resolution for thedifferent Protein C activities is obtained when Mn²⁺ and Mg²⁺ ions areadded to a final concentration of 0.04 mmol/L and 0.4 mmol/L,respectively, thus constituting an improved two-stage method fordetermination of Protein C activity.

Example 4

The effect of manganese ions on the determination of Protein C activityin a two-stage thrombin generation assay using a phospholipid emulsionfrom bovine brain was assessed.

Phospholipid source: Cephotest (Nycomed, Oslo, Norway)

Experimental details are as in Example 3. The final concentration ofCephotest was 3% (v/v).

Results: The results are shown in the table below expressed asOD_(405-490 nm):

Protein C, IU/mL Ions 0 0.1 0.5 Ca²⁺, 6 mmol/L 1.09 0.813 0.375 Ca²⁺,1.5 mmol/L + Mn²⁺, 0.04 mmol/L 1.366 0.996 0.163

The results show that the same enhancing effect on the Protein Canticoagulant activity is obtained with a crude phospholipid extractfrom an animal tissue source. Hence, the source of phospholipid is notcritical.

Example 5

The effect of manganese and magnesium ions on determination of freeProtein S activity in a chromogenic Factor Xa generation assay wasassessed.

Sample: Protein S deficient plasma (Biopool AB) with or without additionof purified human Protein S to yield 0%, 25% and 100% normal Protein Sactivity.

Sample dilution: 1:61 in 50 mmol/L Tris buffer pH 8.2, 0.15 mol/L NaCl,0.2% BSA.

Factor reagent: (concentration in assay before substrate addition):

Bovine FIXa (4 mU/mL)

Bovine FX (0.3 U/mL)

Human FVIII (0.02 U/mL)

Human FV (0.02 U/mL)

Human prothrombin (0.01 U/mL)

Phospholipids (21 μmol/L)

Mg²⁺ (0.4 mmol/L) or Mn²⁺ (0.04 mmol/L) or no addition

Medium: 10 mmol/L MES pH 6.0, 0.15 mol/L NaCl, 0.2% BSA

Start reagent:

Human APC (0.35 μg/mL)

CaCl₂ (1.5 mmol/L)

Chromogenic Factor Xa substrate: S-2765 (Chromogenix AB), 1.8 mmol/L.

To carry out the assay, 50 μL of the diluted plasma sample was mixedwith 50 μL of the above Factor reagent, whereafter the mixture wasincubated for three minutes at 37° C. Thereafter, 50 μL of the Startreagent comprising human APC and calcium chloride was added and themixture was incubated for four minutes at 37° C. Following that, 50 μLof the chromogenic substrate S-2765 was added and the reaction mixtureincubated for two minutes at 37° C., whereafter 50 μL acetic acid wasadded to terminate the reaction. The absorbance of the sample was thendetermined according to Example 1 and expressed as OD_(405-490 nm).

Assay: Factor reagent 50 μL Sample dilution 50 μL Incubation 3 min, 37°C. APC/CaCl₂ 50 μL 4 min, 37° C. S-2765, 1.8 mmol/L 50 μL 2 min, 37° C.HOAc, 20% 50 μL

Results: All primary data are listed in the table below and alsoillustrated in FIG. 3.

Free Protein S, % Ions 0 25 100 Ca²⁺, 1.5 mmol/L 0.857 0.642 0.364 Ca²⁺,1.5 mmol/L + Mn²⁺, 0.04 mmol/L 1.53 1.34 0.745 Ca²⁺, 1.5 mmol/L + Mg²⁺,0.4 mmol/L 1.053 0.851 0.378

FIG. 3 shows that the addition of Mg²⁺ or Mn²⁺ ions results in a greaterresolution (i.e., a greater slope of the curve), as well as in a morelinear dose response when compared to the use of Ca²⁺ alone, thusconstituting an improved method for determination of Protein S activity.

Example 6

The effect of strontium ions on the determination of free Protein Sactivity in a chromogenic Factor Xa generation assay was assessed.

The experimental details are as disclosed in Example 5 but with theFactor reagent stored for one hour before assay.

Results: The results are shown in the table below expressed asOD_(405-490 nm):

Free Protein S, % Ions 0 25 100 Ca²⁺, 1.5 mmol/L 0.673 0.488 0.258 Ca²⁺,1.5 mmol/L + Sr²⁺, 0.4 mmol/L 0.848 0.611 0.276

The results show that a higher resolution for various Protein S activitylevels is obtained on addition of Sr²⁺ ions, supporting the enhancingeffect of Sr²⁺ on the Protein C anticoagulant pathway activity.

Example 7

The effect of metal ions on the detection of Protein S deficiency in aglobal chromogenic method for the Protein C anticoagulant pathway, usingtissue factor as activator of coagulation and monitoring thrombingeneration, was assessed.

Samples: Human pooled normal plasma and Protein S deficient plasma(Biopool AB).

Sample dilution: 1:21 in 25 mmol/L Tris-HCl, pH 7.6, 20 mmol/L NaCl,0.2% bovine serum albumin.

Protein C activator: Protein C activator (Protac®C) from CoamaticProtein C kit (Chromogenix AB) was reconstituted in 7.2 mL according tothe package insert and then diluted in 25 mmol/L Tris-HCl, pH 7.6, 20mmol/L NaCl, 0.2% bovine serum albumin to yield a concentration duringProtein C activation of 0.02 U/mL. Human prothrombin (Chromogenix AB)was added to yield a final concentration after addition of tissue factorof 1.5 μg/mL.

The analysis was performed with or without Mg²⁺ ions added to theProtac®C solution.

Tissue factor: Thromborel (Behringwerke, Marburg, Germany).Reconstituted in 2 mL water according to the manufacturer'sinstructions, thereafter diluted in 25 mmol/L Tris-HCl, pH 7.6, 20mmol/L NaCl, 0.2% bovine serum albumin to yield a final concentrationduring activation of coagulation of 0.033% (v/v).

Phospholipids: 43% phosphatidylcholine, 27% phosphatidylserine and 30%sphingomyelin (Chromogenix AB). Final concentration during activation ofcoagulation of 16.7 μmol/L.

CaCl₂: 6.6 mmol/L final concentration during activation of coagulation.

Chromogenic thrombin substrate: S-2796 (Chromogenix AB), 1.8 mmol/L.

To carry out the assay, 50 μL of the diluted plasma sample was mixedwith 50 μL of the Protein C activator, whereafter the mixture wasincubated for two minutes at 37° C. Thereafter, 50 μL of the reagentcomprising the tissue factor was added and the mixture was incubated fortwo minutes at 37° C. Following that, 50 μL of the chromogenic substrateS-2796 was added and the reaction mixture incubated for four minutes at37° C., whereafter 50 μL acetic acid solution was added to terminate thereaction. The absorbance of the sample was then determined according toExample 1 and expressed as OD_(405-490 nm).

Microplate Assay: Sample dilution 50 μL Protac C activator 50 μL 2 min,37° C. Reagent 50 μL 2 min, 37° C. S-2796 50 μL 4 min, 37° C. HOAc, 20%50 μL

Results: The results are shown in the table below.

Ions Normal plasma Protein S def. plasma Ca²⁺, 6.6 mmol/L 0.26 0.53Ca²⁺, 6.6 mmol/L + Mg²⁺, 0.4 mmol/L 0.29 0.75

The results show that the addition of magnesium ions to calcium ionsbrings about a higher resolution at different Protein S activity levels,thus improving detection of Protein S deficiency.

Example 8

The effect of metal ions on the resolution between different levels offree Protein S, and for detection of FV:Q⁵⁰⁶, in a global method for theProtein C anticoagulant pathway, using Factor Xa as activator ofcoagulation and monitoring thrombin generation, was assessed.

Experimental details are as in Example 7, but with bovine Factor Xa(Chromogenix AB) used instead of tissue factor as activator ofcoagulation. Concentration of Factor Xa=1.4 ng/mL during activation.Furthermore, a stock solution of Protac®C, containing 10 U/mL, was used,which was then diluted in 25 mmol/L Tris-HCl, pH 8.4, 0.2% bovine serumalbumin to yield a concentration during protein C activation of 0.02U/mL.

Samples: 100% protein S = human pooled normal plasma  0% protein S =protein S deficient plasma  25% protein S = protein S deficient plasma +2.5 μg/mL purified human protein S.

Furthermore, a sample from an individual with heterozygosity for thefactor V mutation (FV:R506Q) was analyzed.

Results: See FIG. 4. The table below presents all primary data expressedas OD_(405-490 nm).

Ca²⁺ + Ca²⁺ + Sample Only Ca²⁺ 0.04 mM Mn²⁺ 0.4 mM Mg²⁺ 100% Protein S0.222 0.309 0.414  25% Protein S 0.411 0.485 0.667  0% Protein S 0.6500.975 1.147 FV:R506Q 0.402 0.693 0.761

The results show that a higher resolution is obtained for Protein Sdeficiency in the 0-100% range as well as a high discrimination for theFV: Q⁵⁰⁶ mutation when Mg²⁺ or Mn²⁺ ions are included in the reactionmixture, thus proving the beneficial use of added metal ions in a globalchromogenic method.

Example 9

A comparison was made between a global chromogenic method according tothe invention, using Factor Xa as coagulation activator, with a globalclotting method according to the prior art, using an APTT reagent ascoagulation activator, regarding the resolution between different levelsof Protein C and Protein S activity, and regarding the analysis ofplasma from pregnant women.

Samples: Human pooled normal plasma (PNP), three plasmas from healthyindividuals (N1-N3), and four plasmas from pregnant women (P1-P4) weretested. In addition, plasmas with 0% and 50% deficiency of Protein C (0%Pr C and 50% PrC, respectively), and plasmas with 0% and 50% deficiencyof Protein S (0% Pr S and 50% Pr S, respectively), were tested. The 50%Pr C and PR S plasmas were prepared by adding purified human Protein C(Chromogenix AB) or Protein S (Chromogenix AB) to plasmas deficient ineither Protein C or S (both from Biopool AB).

Global chromogenic assay: Experimental details and assay are as inExamples 7 and 8. A stock solution of Protac®C, initially containing 10U/mL, was diluted to yield a final concentration during activation ofProtein C=0.02 U/mL. The additional metal ion employed was Mn²⁺, whichwas added to the Protac®C solution to yield 0.04 mmol/L in the Protein Cactivation step. The analysis was performed in a microplate, and theOD₄₀₅₋₄₉₀ was determined as described in Example 1. A high OD₄₀₅₋₄₉₀corresponds to pronounced thrombin formation and thus an impairedProtein C anticoagulant pathway activity.

Global clotting assay using APTT reagent: APTT reagent from Coatest® APCResistance kit (Chromogenix AB) was used at a final phospholipidconcentration of 33 μmol/L during coagulation activation. For activationof Protein C, the same Protac®C stock solution and dilution medium wasused as for the chromogenic assay. The final concentration duringactivation of Protein C was 0.083 U/mL. The analysis was performed in aST-4 coagulation analyzer (Diagnostica Stago).

Assay: Plasma sample 50 μL Protac ® C or buffer 50 μL APTT reagent 50 μLActivation for 3 min, 37° C. Ca²⁺, 25 mmol/L 50 μL

The clotting time in seconds was determined in the presence (CT+) andabsence (CT−) of Protac®C and a clot time ratio (CTR) was calculated asCTR=CT+/CT−. A low CTR corresponds to pronounced thrombin formation evenin the presence of Protac®C, and hence an impaired Protein Canticoagulant pathway activity.

Results: The results are shown in the table below.

Chromogenic APTT Sample OD₄₀₅₋₄₉₀ CTR PNP 0.202 3.77 N1 0.183 5.17 N20.182 3.50 N3 0.186 4.85 P1 0.221 3.15 P2 0.298 2.21 P3 0.239 2.60 P40.259 2.66 50% Pr S 0.578 3.48  0% Pr S 0.802 1.80 50% Pr C 0.456 3.86 0% Pr C 1.084 1.18

The results demonstrate that (a) for samples with 50% deficiency ofeither Protein C or Protein S, a higher resolution was obtained versusthe normal samples and (b) for samples from pregnant women, a smallerdeviation from normal samples is obtained with the chromogenic assay,thus supporting the conclusion that a higher sensitivity and specificitywill be obtained with a global chromogenic assay according to theinvention as compared to a global clotting method according to the priorart.

Example 10

The effect of a mixture of Mg²⁺ and Mn²⁺ in a phospholipid reagent or inan APC reagent on the discrimination of the FV:Q⁵⁰⁶ mutation in achromogenic thrombin generation assay, using Factor Xa as activator, wasassessed.

Sample: Plasmas with normal Factor V (R506R), and with heterozygosity(R506Q) and homozygosity (Q506Q) for FV:Q⁵⁰⁶ mutation.

Sample dilution: 1:41 in 0.05 mol/L HEPES, pH 7.7, 0.15 mol/L NaCl.

Reagent A:

Human prothrombin, 19 μg/mL

Phospholipids (43% phosphatidylcholine, 27% phosphatidylserine and 30%sphingomyelin), 50 μmol/L

Reagent B:

Bovine Factor Xa, 0.2 nmol/L

APC, 6 μg/mL

CaCl₂, 25 mmol/L

Chromogenic thrombin substrate: S-2796 (Chromogenix AB), 1.8 mmol/L

Mixture of metal ions: Mg²⁺, 0.4 mmol/L, and Mn²⁺, 0.04 mmol/L, includedin either Reagent A or Reagent B.

To carry out the assay, 50 μL of Reagent A was mixed with 50 μL ofReagent B, whereafter the mixture was incubated for three minutes at 37°C. Thereafter, 50 μL of the plasma dilution was added and incubated fortwo minutes at 37° C. Following that, 50 μL of the chromogenic substrateS-2796 was added and kinetic analysis was performed. The change inOD₄₀₅₋₄₉₀ per minute was determined and expressed as ΔOD₄₀₅₋₄₉₀/min.

Assay: Reagent A 50 μL Reagent B 50 μL Incubate at 37° C. for 3 minPlasma dilution 50 μL 2 min, 37° C. S-2796 50 μL Kinetic reading

Results: The results are shown in the table below.

Mg²⁺ and Mn²⁺ Mg²⁺ and Mn²⁺ in Reagent A in Reagent B FV:R506R 0.1430.193 FV:R506Q 0.646 0.616 FV:Q506Q 1.116 0.942

The results show that a mixture of metal ions, such as Mg²⁺ and Mn²⁺,may be added in a phospholipid containing reagent (Reagent A), or in areagent containing active enzymes such as APC and Factor Xa (Reagent B),and can provide a high discrimination for the FV:Q⁵⁰⁶ mutation. Hencethe addition of additional metal ions is not restricted to any uniquereagent.

Example 11

The substitution of chloride anions with nitrate and sulfate anions wasassessed in a study on the effect of magnesium and manganese indetermination of Protein C activity in a three-stage thrombin generationassay.

Experimental details are as in Example 1, except that magnesium nitrate(Mg(NO₃)₂) and manganese sulfate (MnSO₄) were used instead of thecorresponding chloride salts at final concentrations in the assay of 0.4and 0.04 mmol/L, respectively, in accordance with the conditions inExample 1.

Results: The results are shown in the table below expressed asOD_(405-490 nm).

Protein C, IU/mL Ions 0 0.1 0.5 1.0 Ca²⁺, 1.5 mmol/L + 0.563 0.541 0.2780.079 Mn²⁺, 0.04 mmol/L Ca²⁺, 1.5 mmol/L + 0.603 0.554 0.448 0.154 Mg²⁺,0.4 mmol/L

The results show that a similar high resolution is obtained as whenusing chloride as an anion (cf. Example 1). Thus, the choice of theanion is not restricted to chloride ions.

Example 12

The effect of metal ions on the detection of Protein C deficiency,Protein S deficiency, and the FV:Q⁵⁰⁶ mutation was assessed in a globalchromogenic method for the Protein C anticoagulant pathway, usingrecombinant tissue factor as activator of coagulation and monitoringthrombin generation.

Experimental details are as in Example 7, but using recombinant tissuefactor (PT-Fibrinogen Recombinant, Instrumentation Laboratory, Milano,Italy) instead of Thromborel as activator of coagulation. PT-FibrinogenRecombinant was reconstituted with 8 mL of water according to themanufacturer's instructions, thereafter diluted in 25 mmol/L Tris-HCl,pH 7.6, 20 mmol/L NaCl, 0.2% bovine serum albumin to yield a finalconcentration during activation of coagulation of 0.25% (v/v).

Samples: Normal human plasma, Protein C deficient plasma, and Protein Sdeficient plasma (Instrumentation Laboratory, Milano, Italy). Plasmaswith 25% activity of Protein C and Protein S, respectively, wereprepared by mixing normal human plasma with the Protein C or Protein Sdeficient plasmas respectively. Furthermore, a sample from an individualwith heterozygosity for the factor V mutation (FV:R506Q) and from anindividual with homozygosity for the same mutation (FV:Q506Q) wereanalyzed.

The analysis was performed with or without Mg²⁺ ions added to theProtein C activator solution.

Results: See FIG. 5. The table below presents all primary data expressedas OD_(405-490 nm).

6.6 mM Ca²⁺ + Sample 6.6 mM Ca²⁺ 0.4 mM Mg²⁺ Normal Plasma 0.881 0.36425% Protein C 1.225 1.047  0% Protein C 1.418 1.388 25% Protein S 1.3250.837  0% Protein S 1.456 1.421 FV:R506Q 1.140 0.686 FV:Q506Q 1.3921.398

The results show that the presence of magnesium ions during the ProteinC activation and during the ensuing thrombin generation provides anenhancement of the anticoagulant activity (see results for normalplasma). Furthermore, the enhanced anticoagulant activity results in ahigher resolution at different Protein C and Protein S activity levels,as well as higher discrimination for the FV:Q⁵⁰⁶ mutation.

Example 13

The effect of manganese ions on the discrimination at different ProteinS activity levels was assessed in a global chromogenic method for theProtein C anticoagulant pathway, using tissue factor as activator ofcoagulation and monitoring thrombin generation.

Experimental details are as in Example 7, using Protac® C as the ProteinC activator and Thromborel as activator of coagulation.

Samples: Normal human plasma (Instrumentation Laboratory, Milano, Italy)was used as the 100% Protein S sample; and Protein S deficient plasma(Instrumentation Laboratory, Milano, Italy) was used as the 0% Protein Ssample. Additional plasma samples were prepared by mixing normal humanplasma and Protein S deficient plasma to yield plasmas with 20%, 40%,60% and 80% Protein S activity respectively.

The analysis was performed with or without Mn²⁺ ions added to theProtein C activator solution.

Results: All primary data expressed as OD_(405-490 nm) are listed in thetable below and are also illustrated in FIG. 6.

6.6 mM Ca²⁺ + Sample 6.6 mM Ca²⁺ 0.04 mM Mn²⁺  0% Protein S 0.458 1.371 20% Protein S 0.407 0.756  40% Protein S 0.388 0.570  60% Protein S0.367 0.493  80% Protein S 0.348 0.439 100% Protein S 0.325 0.379

FIG. 6 shows that the addition of Mn²⁺ ions dramatically increases theresolution when compared to the use of Ca²⁺ alone, thus constituting animproved detection of Protein S deficiency in a global chromogenicmethod for detection of deficiency states of components in the Protein Canticoagulant pathway.

Example 14

The effect of magnesium and manganese ions on the discrimination atdifferent Protein C activity levels was assessed in a global chromogenicmethod for the Protein C anticoagulant pathway, using recombinant tissuefactor as the activator of coagulation, a recombinant Protein Cactivator, and monitoring thrombin generation.

Experimental details are as in Example 7, but using recombinant ProteinC activator as Protein C activator and recombinant tissue factor(PT-Fibrinogen Recombinant, Instrumentation Laboratory) as activator ofcoagulation. Recombinant Protein C activator was used as a stocksolution containing 26 U/mL. The recombinant Protein C activator wasthen diluted in 25 mmol/L Tris-HCl, pH 7.6, 20 mmol/L NaCl, 0.2% bovineserum albumin to yield a final concentration during activation ofProtein C of 0.025 U/mL. PT-Fibrinogen Recombinant was prepared as inExample 12 to yield a concentration during activation of coagulation of0.17% (v/v).

Samples: Normal human plasma (Instrumentation Laboratory, Milano, Italy)was used as the 100% Protein C sample; Protein C deficient plasma(Instrumentation Laboratory, Milano, Italy) was used as the 0% Protein Csample; and additional plasma samples were prepared by mixing normalhuman plasma and Protein C deficient plasma to yield plasmas with 20%,40%, 60% and 80% Protein C activity respectively.

The analysis was performed with or without Mg²⁺ or Mn²⁺ ions added tothe recombinant Protein C activator solution.

Results: All primary data expressed as OD_(405-490 nm) are listed in thetable below and also illustrated in FIG. 7.

6.6 mM Ca²⁺ + 6.6 mM Ca²⁺ + Sample 6.6 mM Ca²⁺ 0.4 mM Mg²⁺ 0.4 mM Mn²⁺ 0% Protein C 1.442 1.483 1.451 20% Protein C 1.253 1.219 1.097 40%Protein C 1.010 0.900 0.636 60% Protein C 0.783 0.675 0.438 80% ProteinC 0.787 0.569 0.356 100% Protein C  0.672 0.456 0.280

FIG. 7 shows that the addition of Mg²⁺ or Mn²⁺ ions results in a higherresolution for the different Protein C activities when compared to theuse of Ca²⁺ alone, thus resulting in an improved detection of Protein Cdeficiency in a global chromogenic method for detection of deficiencystates of components in the Protein C anticoagulant pathway.

Example 15

The effect of the combination of Mg²⁺ and Mn²⁺ ions on the detection ofProtein C deficiency, Protein S deficiency and the FV:Q⁵⁰⁶ mutation wasassessed in a global chromogenic method for the Protein C anticoagulantpathway, using tissue factor as activator of coagulation and monitoringthrombin generation.

Experimental details are as in Example 7, using Protac® C as the ProteinC activator and Thromborel as the activator of coagulation.

Samples: Normal human plasma, Protein C deficient plasma and Protein Sdeficient plasma (Instrumentation Laboratory, Milano, Italy).Furthermore, a sample from an individual heterozygous for the Factor Vmutation (FV:R506Q) and from an individual homozygous for the samemutation (FV:Q506Q) were analyzed.

The analysis was performed with or without the presence of thecombination of Mg²⁺ and Mn²⁺ ions added to the Protein C activatorsolution.

Results: See FIG. 8. The table below presents all primary data expressedas ΔAbnormal—Normal (i.e., OD₄₀₅₋₄₉₀ abnormal plasma—OD₄₀₅₋₄₉₀ normalplasma).

6.6 mM Ca²⁺ + 0.4 mM Mg²⁺ + Sample 6.6 mM Ca²⁺ 0.04 mM Mn²⁺ 0% Protein C0.425 0.801 0% Protein S 0.372 0.742 FV:R506Q 0.060 0.413 FV:Q506Q 0.5300.664

The results show that the addition of the combination of magnesium andmanganese ions to calcium ions provide a higher resolution for bothProtein C and Protein S deficiencies, as well as a higher discriminationfor the FV:Q⁵⁰⁶ mutation, thus proving the beneficial use of adding acombination of metal ions in a global chromogenic method.

What is claimed is:
 1. A method for determining the activity of one ormore components of a Protein C anticoagulant pathway, the methodcomprising the steps of: (a) adding a procoagulant reagent to a bloodsample to activate a coagulation cascade; (b) adding calcium ions to theblood sample to trigger coagulation; (c) adding at least one metal ionto the blood sample at a concentration that increases anticoagulantactivity of the Protein C anticoagulant pathway, the metal ion selectedfrom the group consisting of Mg⁺², Mn⁺², Zn⁺², Ni⁺², Sr⁺², Cu⁺², andCu⁺; (d) adding an exogenous substrate for an enzyme influenced byProtein C anticoagulant activity; and (e) comparing a characteristic ofthe exogenous substrate with a characteristic of the exogenous substrateas determined by the method recited in steps (a)-(d) for a normal bloodsample.
 2. The method according to claim 1, wherein the metal ioncomprises Mg⁺² and is added to a final concentration of about 20 μmol toabout 10 mmol per liter.
 3. The method according to claim 1, wherein themetal ion comprises Mg⁺² and is added to a final concentration of about100 μmol to about 2 mmol per liter.
 4. The method according to claim 1,wherein the metal ion comprises Mg⁺² and is added to a finalconcentration of about 200 μmol to about 1 mmol per liter.
 5. The methodaccording to claim 1, wherein the metal ion comprises at least one ofMn⁺², Zn⁺², Ni⁺², Sr⁺², Cu⁺², and Cu⁺, and is added to a finalconcentration of about 1 μmol to about 2 mmol per liter.
 6. The methodaccording to claim 1, wherein the metal ion comprises at least one ofMn⁺², Zn⁺², Ni⁺², Sr⁺², Cu⁺², and Cu⁺, and is added to a finalconcentration of about 5 μmol to about 400 μmol per liter.
 7. The methodaccording to claim 1, wherein the metal ion comprises at least one ofMn⁺², Zn⁺², Ni⁺², Sr⁺², Cu⁺², and Cu⁺, and is added to a finalconcentration of about 10 μmol to about 80 μmol per liter.
 8. The methodaccording to claim 1, wherein the exogenous substrate comprises asubstrate for a component of the coagulation cascade selected from thegroup consisting of Factor Xa and thrombin.
 9. The method according toclaim 8, wherein the substrate is selected from the group consisting ofBenzoyl-Ile-Glu-Gly-Arg-pNA, N-a-Z-D-Arg-Gly-Arg-pNA,CH₃SO₂-D-Leu-Gly-Arg-pNA, and MeO-CO-D-CHG-Gly-Arg-pNA.
 10. The methodaccording to claim 8, wherein the substrate is selected from the groupconsisting of H-D-Phe-Pip-Arg-pNA, pyroGlu-Pro-Arg-pNA,H-D-Ala-Pro-Arg-pNA, Z-D-Arg-Sarc-Arg-pNA, AcOH*H-D-CGH-But-Arg-pNA andH-D-HHT-Ala-Arg-pNA.
 11. The method according to claim 1, wherein theexogenous substrate comprises a photometrically measurable leavinggroup.
 12. The method according to claim 11, wherein the photometricallymeasurable leaving group is selected from the group consisting of achromophore, a fluorophore, and a luminophore.
 13. The method accordingto claim 12, wherein the chromophore comprises a p-nitroaniline group(pNA).
 14. The method according to claim 12, wherein the fluorophorecomprises a naphthylamine or coumarine derivative group.
 15. The methodaccording to claim 12, wherein the luminophore comprises anisoluminolamide group.
 16. The method according to claim 1, wherein theblood sample is selected from the group consisting of whole blood, bloodplasma, and blood serum.
 17. The method according to claim 1, whereinsteps (a) through (d) are performed separately.
 18. The method accordingto claim 1, wherein steps (a) through (d) are performed simultaneously.19. The method according to claim 1, wherein the calcium ions are addedto a final concentration of about 0.5 mmol to about 20 mmol per liter.20. The method according to claim 1, wherein the calcium ions are addedto a final concentration of about 1 mmol to about 10 mmol per liter. 21.The method according to claim 1, wherein the procoagulant reagentcomprises at least one of a phospholipid and a contact activator. 22.The method according to claim 1, wherein the procoagulant reagentcomprises at least one phospholipid and at least one intrinsic pathwayfactor selected from the group consisting of Factor IXa, Factor XIIa,and Factor XIa.
 23. The method according to claim 1, wherein theprocoagulant reagent comprises at least one phospholipid, at least onecontact activator, and at least one intrinsic pathway factor selectedfrom the group consisting of Factor IXa, Factor XIIa, and Factor XIa.24. The method according to any one of claims 21-23, wherein the atleast one phospholipid is selected from the group consisting of asynthetic phospholipid, a purified phospholipid, and a crude extract ofa phospholipid derived from a biological source.
 25. The methodaccording to claim 23, wherein the at least one contact activator isselected from the group consisting of ellagic acid, collagen, acollagen-related substance, and silica.
 26. The method according toclaim 25, wherein the silica is selected from the group consisting ofmicronized silica, colloidal silica, and kaolin.
 27. The methodaccording to claim 1, wherein the procoagulant reagent comprises atleast one material selected from the group consisting of native humantissue factor, recombinant human tissue factor, native non-human tissuefactor, recombinant non-human tissue factor, native human FactorVII/Factor VIIa, recombinant human Factor VII/Factor VIIa, nativenon-human Factor VII/Factor VIIa, and recombinant non-human FactorVII/Factor VIIa.
 28. The method according to claim 24, wherein the atleast one phospholipid is selected from the group consisting ofphosphatidylcholine, phosphatidylserine, and sphingomyelin.
 29. Themethod according to claim 1, wherein theprocoagulant reagent comprises amaterial selected from the group consisting of exogenous Factor Xa,exogenous Factor X and an exogenous activator of Factor X, and anexogenous activator for endogenous Factor X.
 30. The method according toclaim 29, wherein the exogenous activator for Factor X comprises snakevenom enzyme.
 31. The method according to claim 30, wherein the snakevenom enzyme comprises Russelli Viperii snake venom enzyme.
 32. Themethod according to claim 1, the method further comprising the step ofadding to the blood sample at least one component selected from thegroup consisting of Protein C, activated Protein C, Protein S, Factor V,and Factor Va.
 33. The method according to claim 1, wherein a fibrinpolymerization inhibitor is added to the blood sample.
 34. The methodaccording to claim 33, wherein the fibrin polymerization inhibitorcomprises Gly-Pro-Arg-Pro.
 35. The method according to claim 1, whereinthe procoagulant reagent comprises at least one material selected fromthe group consisting of Factor VIII/Factor VIIIa, Factor X, andprothrombin.
 36. The method according to claim 1, the method furthercomprising the step of providing activated Protein C by adding exogenousactivated Protein C to the blood sample.
 37. The method according toclaim 1, the method further comprising the step of providing activatedProtein C by adding an activator of Protein C to the blood sample. 38.The method according to claim 1, the method further comprising the stepof providing activated Protein C by adding exogenous Protein C and anactivator of Protein C to the blood sample.
 39. The method according toany one of claims 36-38, wherein step (c) occurs simultaneously with theproviding activated Protein C step.
 40. The method according to any oneof claims 36-38, wherein the providing activated Protein C step occurssimultaneously with step (a).
 41. The method according to any one ofclaims 36-38, wherein the providing activated Protein C step precedesstep (a).
 42. The method according to any one of claims 37-38, whereinthe activator of Protein C comprises at least one of Protein Cactivating snake venom enzyme and thrombin.
 43. The method according toany one of claims 37-38, wherein the activator of Protein C comprisesthrombomodulin and thrombin.
 44. The method according to any one ofclaims 37-38, wherein the activator of Protein C is recombinant.
 45. Themethod according to claim 42, wherein the snake venom enzyme is derivedfrom the Agkistrodon family.
 46. The method according to claim 45,wherein the snake venom enzyme is derived from Agkistrodon contortrixcontortrix.
 47. The method according to claim 45, wherein the snakevenom enzyme comprises crude snake venom enzyme.
 48. The methodaccording to claim 45, wherein the snake venom enzyme comprises purifiedsnake venom enzyme.
 49. The method according to claim 48, wherein thetotal amount of purified snake venom enzyme is about 1×10⁻³ U to about 1U per milliliter.
 50. The method according to claim 48, wherein thetotal amount of purified snake venom enzyme is about 2×10 ⁻³ U to about0.3 U per milliliter.
 51. The method according to claim 1, wherein thecharacteristic comprises a conversion rate.
 52. The method according toclaim 51, wherein the conversion rate is measured kinetically.
 53. Themethod according to claim 51, wherein the conversion rate is measuredafter a fixed incubation time.
 54. The method according to claim 1,wherein the procoagulant reagent comprises Factor VIIa and at least onephospholipid.
 55. The method according to claim 1, wherein theprocoagulant reagent comprises at least one material selected from thegroup consisting of prothrombin, Factor V/Va, Factor IX, and Factor X.56. The method according to claim 1, the method further comprising thestep of adding to the blood sample a plasma deficient in at least onecomponent selected from the group consisting of Protein C, Protein S,and Factor V.
 57. The method according to claim 1, the method furthercomprising the step of adding to the blood sample a plasma deficient inall components of the Protein C anticoagulant pathway.
 58. The methodaccording to claim 1, the method further comprising the step of addingto the blood sample a plasma deficient in the Protein C anticoagulantpathway component to be measured.
 59. The method according to any one ofclaims 36-38, wherein each step is performed separately.
 60. The methodaccording to any one of claims 36-38, wherein two or more steps areperformed simultaneously.
 61. The method according to any one of claims36-38, wherein the providing activated Protein C step and step (a) areperformed simultaneously and are followed by simultaneous performance ofstep (b) and step (d).
 62. The method according to any one of claims36-38, wherein the providing activated Protein C step, step (a), andstep (d) are performed simultaneously and are followed by performance ofstep (b).
 63. The method according to any one of claims 36-38, whereinthe providing activated Protein C step, step (a), and step (b) areperformed simultaneously and are followed by performance of step (d).64. The method according to any one of claims 36-38, wherein theproviding activated Protein C step and step (a) are performedsimultaneously and are followed by performance of step (d) andperformance of step (b).
 65. The method according to anyone of claims36-38, wherein the providing activated Protein C step and step (a) areperformed separately and step (d) and step (b) are performedsimultaneously.
 66. The method according to any one of claims 36-38,wherein the providing activated Protein C step and step (b) areperformed simultaneously and step (a) and step (d) are performedseparately.
 67. The method according to any one of claim 36-38, whereinthe providing activated Protein C step is performed separately, step (a)and step (b) are performed simultaneously, and step (d) is performedseparately.
 68. The method according to claim 1, further comprising theaddition of a counter ion selected from the group consisting of amonovalent anion, a divalent anion, and a trivalent anion.
 69. Themethod according to claim 68, wherein the counter ion is an anionselected from the group consisting of chloride, sulfate, and nitrate.